Epigenetic modifications provide essential mechanisms by which cells containing identical genomes can adopt and maintain different cell identities. These mechanisms are crucial for maintaining pools of stem cells that can differentiate and replenish tissue lost during homeostasis, injury, and aging. To maintain the stem cell pool, asymmetric cell division (ACD) occurs where one daughter cell becomes a self-renewed stem cell and the other daughter goes on to differentiate. Disruption of this balance can be disastrous, leading to defects ranging from cancer to degenerative diseases. Despite its importance, little is known about which and how the epigenetic information of the stem cell could be distributed between its two daughters during ACD. The project proposed here will (1) permit visualization of the epigenetic information contained at gene-specific loci on segregating sister chromatids during ACD and (2) describe the cis and trans elements involved in the faithful segregation of the epigenetically distinct chromatids between the two daughter cells. A major player in epigenetic control is the nucleosome, an octamer of histone proteins that intimately interacts with DNA and has N-terminal `tails' that can be extensively modified to affect gene expression. It is known that the age of a histone is correlated with the types of epigenetic modifications it carries. Thus, it may be possible that the daughters of an ACD may acquire their different fates by inheriting `old' versus `new' histones at genes important for maintaining the stem cell state or promoting differentiation. The Drosophila germline permits visualization of asymmetric germline stem cell (GSC) division at single-cell resolution in vivo, making it a great system to study histone segregation between the daughters of an ACD. Current data in the lab has indeed found significant regions of non-overlapping old and new histone signals in mitotic GSCs. To investigate these regions, a new imaging technique has been developed. In short, fluorescent probes targeting loci containing either `stemness' or differentiation genes are applied to determine if a particular gene is preferentially associated with old versus new histones. Further, these probes will be used with antibodies against specific histone modifications to reveal the epigenetic differences between the renewed stem cell daughter and the daughter destined to differentiate in ACD. Live and fixed imaging will elucidate the dynamics of both cis elements on the chromatids and trans regulatory factors on the mitotic machinery to determine the roles they play in sister chromatid recognition and segregation during GSC ACD. The results will reveal how gene-specific epigenetic control is established during ACD as well as provide a spatiotemporal map of the molecular machinery and cellular events involved in faithfully segregating that information. These methods can be widely applied to other organisms and cell lineages, which will enhance the capacity to study the crucial process of ACD. The data and techniques generated here will significantly impact fundamental knowledge in the fields of stem cell and chromatin biology.